Chimeric genes suitable for expression in plant cells

ABSTRACT

This invention relates to chimeric genes which are capable of being expressed in plant cells. Such genes contain (a) a promoter region derived a gene which is expressed in plant cells, such as the nopaline synthase gene; (b) a coding or structural sequence which is heterologous with respect to the promoter region; and (c) an appropriate 3′ non-translated region. Such genes have been used to create antibiotic-resistant plant cells; they are also useful for creating herbicide-resistant plants, and plants which contain mammalian polypeptides.

This is a Divisional of U.S. application Ser. No. 08/127,100, filed Sep.24, 1993, which is a File Wrapper Continuation of U.S. application Ser.No. 07/732,974, filed Jul. 19, 1991, now abandoned, which is aContinuation of application Ser. No. 07/333,802, filed Apr. 5, 1989, nowU.S. Pat. No. 5,034,322, which is a Continuation of U.S. applicationSer. No. 06/793,488, filed Oct. 30, 1985, now abandoned, which is aContinuation of U.S. application Ser. No. 06/458,414 filed Jan. 17,1983, now abandoned.

TECHNICAL FIELD

This invention is in the fields of genetic engineering, plant biology,and bacteriology.

BACKGROUND ART

In the past decade, the science of genetic engineering has developedrapidly. A variety of processes are known for inserting a heterologousgene into bacteria, whereby the bacteria become capable of efficientexpression of the inserted genes. Such processes normally involve theuse of plasmids which may be cleaved at one or more selected cleavagesites by restriction endonucleases, discussed below. Typically, a geneof interest is obtained by cleaving one piece of DNA and the resultingDNA fragment is mixed with a fragment obtained by cleaving a vector suchas a plasmid. The different strands of DNA are then connected(“ligated”) to each other to form a reconstituted plasmid. See, forexample, U.S. Pat. No. 4,237,224 (Cohen and Boyer, 1980); U.S. Pat. No.4,264,731 (Shine, 1981); U.S. Pat. No. 4,273,875 (Manis, 1981); U.S.Pat. No. 4,322,499 (Baxter et al., 1982), and U.S. Pat. No. 4,336,336(Silhavy et al., 1982). A variety of other reference works are alsoavailable. Some of these works describe the natural processes wherebyDNA is transcribed into messenger (mRNA) and mRNA is translated intoprotein; see, e.g., Stryer, 1981 (note: all references cited herein,other than patents, are listed with citations after the Examples);Lehninger, 1975. Other works describe methods and products of geneticmanipulation; see, e.g., Maniatis et al., 1982; Setlow and Hollaender,1979.

Most of the genetic engineering work performed to date involves theinsertion of genes into various types of cells primarily bacteria suchas E. coli, various other types of microorganisms such as yeast, andmammalian cells. However, many of the techniques and substances used forgenetic engineering of animal cells and microorganisms are not directlyapplicable to genetic engineering involving plants.

As used herein, the term “plant” refers to a multicellulardifferentiated organism that is capable of photosynthesis, such asangiosperms and multicellular algae. This does not includemicroorganisms, such as bacteria, yeast, and fungi. However, the term“plant cells” includes any cell derived from a plant; this includesundifferentiated tissue such as callus or crown gall tumor, as well asplant seeds, propagules, pollen, and plant embryos.

A variety of plant genes have been isolated, some of which have beenpublished and/or are publicly available. Such genes include the soybeanactin gene (Shah et al., 1982), corn zein (Pederson et al., 1982)soybean leghemoglobin (Hyldig-Nielsen et al., 1982), and soybean storageproteins (Fischer and Goldberg, 1982).

The Reigons of a Gene

The expression of a gene involves the creation of a polypeptide which iscoded for by the gene. This process involves at least two steps: part ofthe gene is transcribed to form messenger RNA, and part of the mRNA istranslated into a polypeptide. Although the processes of transcriptionand translation are not fully understood, it is believed that thetranscription of a DNA sequence into mRNA is controlled by severalregions of DNA. Each region is a series of bases (i.e., a series ofnucleotide residues comprising adenosine (A), thymidine (T), cytidine(C), and guanidine (G)) which are in a desired sequence. Regions whichare usually present in a eucaryotic gene are shown on FIG. 1. Theseregions have been assigned names for use herein, and are brieflydiscussed below. It should be noted that a variety of terms are used inthe literature, which describes these regions in much more detail.

An association region 2 causes RNA polymerase to associate with thesegment of DNA. Transcription does not occur at association region 2;instead, the RNA polymerase normally travels along an intervening region4 for an appropriate distance, such as about 100-300 bases, after it isactivated by association region 2.

A transcription initiation sequence 6 directs the RNA polymerase tobegin synthesis of mRNA. After it recognizes the appropriate signal, theRNA polymerase is believed to begin the synthesis of mRNA an appropriatedistance, such as about 20 to about 30 bases, beyond the transcriptioninitiation sequence 6. This is represented in FIG. 1 by interveningregion 8.

The foregoing sequences are referred to collectively as the promoterregion of the gene.

The next sequence of DNA is transcribed by RNA polymerase into messengerRNA which is not translated into protein. In general, the 5′ end of astrand of mRNA attaches to a ribosome. In bacterial cells, thisattachment is facilitated by a sequence of bases called a “ribosomebinding site” (RBS). However, in eucaryotic cells, no such RBS sequenceis known to exist. Regardless of whether an RBS exists in a strand ofmRNA, the mRNA moves through the ribosome until a “start codon” isencountered. The start codon is usually the series of three bases, AUG;rarely, the codon GUG may cause the initiation of translation. Thenon-translated portion of mRNA located between the 5′ end of the mRNAand the start codon is referred to as the 5′ non-translated region 10 ofthe mRNA. The corresponding sequence in the DNA is also referred toherein as 5′ non-translated region 12. The specific series of bases inthis sequence is not believed to be of great importance to theexpression of the gene; however, the presence of a premature start codonmight affect the translation of the mRNA (see Kozak, 1978).

A promoter sequence may be significantly more complex than describedabove; for example, certain promoters present in bacteria containregulatory sequences that are often referred to as “operators.” Suchcomplex promoters may contain one or more sequences which are involvedin induction or repression of the gene. One example is the lac operon,which normally does not promote transcription of certainlactose-utilizing enzymes unless lactose is present in the cell. Anotherexample is the trp operator, which does not promote transcription ortranslation of certain tryptophan-creating enzymes if an excess oftryptophan is present in the cell. See, e.g., Miller and Reznikoff,1982.

The next sequence of bases is usually called the coding sequence or thestructural sequence 14 (in the DNA molecule) or 16 (in the mRNAmolecule). As mentioned above, the translation of a polypeptide beginswhen the mRNA start codon, usually AUG, reaches the translationmechanism in the ribosome. The start codon directs the ribosome to beginconnecting a series of amino acids to each other by peptide bonds toform a polypeptide, starting with methionine, which always forms theamino terminal end of the polypeptide (the methionine residue may besubsequently removed from the polypeptide by other enzymes). The baseswhich follow the AUG start codon are divided into sets of 3, each ofwhich is a codon. The “reading frame,” which specifies how the bases aregrouped together into sets of 3, is determined by the start codon. Eachcodon codes for the addition of a specific amino acid to the polypeptidebeing formed. The entire genetic code (there are 64 different codons)has been solved; see, e.g., Lehninger, supra, at p. 962. For example,CUA is the codon for the amino acid leucine; GGU specifies glycine, andUGU specifies cysteine.

Three of the codons (UAA, UAG, and UGA) are “stop” codons; when a stopcodon reaches the translation mechanism of a ribosome, the polypeptidethat was being formed disengages from the ribosome, and the lastpreceding amino acid residue becomes the carboxyl terminal end of thepolypeptide.

The region of mRNA which is located on the 3′ side of a stop codon in amonocistronic gene is referred to herein as 3′ non-translated region 18.This region is believed to be involved in the processing, stability,and/or transport of the mRNA after it is transcribed. This region 18 isalso believed to contain a sequence polyadenylation signal 20, which isrecognized by an enzyme in the cell. This enzyme adds a substantialnumber of adenosine residues to the mRNA molecule, to form poly-A tail22.

The DNA molecule has a 3′ non-translated region 24 and a polyadenylationsignal 26, which code for the corresponding mRNA region 18 and signal20. However, the DNA molecule does not have a poly-A tail.Polyadenylation signals 20 (mRNA) and 26 (DNA) are represented in thefigures by a heavy dot.

Gene-Host Incompatibility

The same genetic code is utilized by all living organisms on Earth.Plants, animals, and microorganisms all utilize the same correspondencebetween codons and amino acids. However, the genetic code applies onlyto the structural sequence of a gene, i.e., the segment of mRNA boundedby one start codon and one stop codon which codes for the translation ofmRNA into polypeptides.

However, a gene which performs efficiently in one type of cell may notperform at all in a different type of cell. For example, a gene which isexpressed in E. coli may be transferred into a different type ofbacterial cell, a fingus, or a yeast. However, the gene might not beexpressed in the new host cell. There are numerous reasons why an intactgene which is expressed in one type of cell might not be expressed in adifferent type of cell. See, e.g., Sakaguchi and Okanishi, 1981. Suchreasons include:

1. The gene might not be replicated or stably inherited by the progenyof the new host cell.

2. The gene might be broken apart by restriction endonucleases or otherenzymes in the new host cell.

3. The promoter region of the gene might not be recognized by the RNApolymerases in the new host cell.

4. One or more regions of the gene might be bound by a repressor proteinor other molecule in the new host cell, because of a DNA region whichresembles an operator or other regulatory sequence of the host's DNA.For example, the lac operon includes a polypeptide which binds to aparticular sequence of bases next to the lac promoter unless thepolypeptide is itself inactivated by lactose. See, e.g., Miller andReznikoff, 1982.

5. One or more regions of the gene might be deleted, reorganized, orrelocated to a different part of the host's genome. For example,numerous procaryotic cells are known to contain enzymes which promotegenetic recombination (such as the rec proteins in E. coli; see, e.g.,Shibata et al., 1979) and transposition (see, e.g., The 45th Cold SpringHarbor Symposium on Quantitative Biology, 1981). In addition,naturally-occurring genetic modification can be enhanced by regions ofhomology between different strands of DNA; see, e.g., Radding, 1978.

6. mRNA transcribed from the gene may suffer from a variety of problems.For example, it might be degraded before it reaches the ribosome, or itmight not be polyadenylated or transported to the ribosome, or it mightnot interact properly with the ribosome, or it might contain anessential sequence which is deleted by RNA processing enzymes.

7. The polypeptide which is created by translation of the mRNA coded forby the gene may suffer from a variety of problems. For example, thepolypeptide may have a toxic effect on the cell, or it may beglycosylated or converted into an altered polypeptide, or it may becleaved into shorter polypeptides or amino acids, or it may besequestered within an intracellular compartment where it is notfunctional.

In general, the likelihood of a foreign gene being expressed in a celltends to be lower if the new host cell is substantially different fromthe natural host cell. For example, a gene from a certain species ofbacteria is likely to be expressed by other species of bacteria withinthe same genus. The gene is less likely to be expressed by bacteria of adifferent genus, and even less likely to be expressed by non-bacterialmicroorganisms such as yeast, fungus, or algae. It is very unlikely thata gene from a cell of one kingdom (the three kingdoms are plants,animals, and “protista” (microorganisms)) could be expressed in cellsfrom either other kingdom.

These and other problems have, until now, thwarted efforts to obtainexpression of foreign genes into plant cells. For example, severalresearch teams have reported the insertion of foreign DNA into plantcells; see, e.g., Lurquin, 1979; Krens et al., 1982; Davey et al., 1980.At least three teams of researchers have reported the insertion ofentire genes into plant cells. By use of radioactive DNA probes, theseresearchers have reported that the foreign genes (or at least portionsthereof) were stably inherited by the descendants of the plant cells.See Hernalsteens et al., 1980; Garfinkel et al., 1981; Matzke andChilton, 1981. However, there was no reported evidence that the foreigngenes were expressed in the plant cells.

Several natural exceptions to the gene-host incompatibility barriershave been discovered. For example, several E. coli genes can beexpressed in certain types of yeast cells, and vice-versa. See Beggs,1978; Struhl et al., 1979.

In addition, certain types of bacterial cells, including Agrobacteriumtumefaciens and A. rhizogenes, are capable of infecting various types ofplant cells, causing plant diseases such as crown gall tumor and hairyroot disease. These Agrobacterium cells carry plasmids, designated as Tiplasmids and Ri plasmids, which carry genes which are expressed in plantcells. Certain of these genes code for enzymes which create substancescalled “opines,” such as octopine, nopaline, and agropine. Opines areutilized by the bacteria cells as sources of carbon, nitrogen, andenergy. See, e.g., Petit and Tempe, 1978. The opine genes are believedto be inactive while in the bacterial cells; these genes are expressedonly after they enter the plant cells.

In addition, a variety of man-made efforts have been reported toovercome one or more of the gene-host incompatibility barriers. Forexample, it has been reported that a mammalian polypeptide which isnormally degraded within a bacterial host can be protected fromdegradation by coupling the mammalian polypeptide to a bacterialpolypeptide that normally exists in the host cell. This creates a“fusion protein;” see, e.g., Itakura et al., 1977. As another example,in order to avoid cleavage of an inserted gene by endonucleases in thehost cell, it is possible to either (1) insert the gene into host cellswhich are deficient in one or more endonucleases, or (2) duplicate thegene in cells which cause the gene to be methylated. See, e.g., Maniatiset al., 1981.

In addition, various efforts to overcome gene-host incompatibilitybarriers involve chimeric genes. For example, a structural sequencewhich codes for a mammalian polypeptide, such as insulin, interferon, orgrowth hormone, may be coupled to regulatory sequences from a bacterialgene. The resulting chimeric gene may be inserted into bacterial cells,where it will express the mammalian polypeptide. See, e.g., Guarente etal., 1980. Alternately, structural sequences from several bacterialgenes have been coupled to regulatory sequences from viruses which arecapable of infecting mammalian cells. The resulting-chimeric genes wereinserted into mammalian cells, where they reportedly expressed thebacterial polypeptide. Southern and Berg, 1982; Colbere-Garapin et al.,1981.

Restriction Endonucleases

In general, an endonuclease is an enzyme which is capable of breakingDNA into segments of DNA. An endonuclease is capable of attaching to astrand of DNA somewhere in the middle of the strand, and breaking it. Bycomparison, an exonuclease removes nucleotides, from the end of a strandof DNA. All of the endonucleases discussed herein are capable ofbreaking double-stranded DNA into segments. This may require thebreakage of two types of bonds: (1) covalent bonds between phosphategroups and deoxyribose residues, and (2) hydrogen bonds (A-T and C-G )which hold the two strands of DNA to each other.

A “restriction endonuclease” (hereafter referred to as an endonuclease)breaks a segment of DNA at a precise sequence of bases. For example,EcoRI and HaeIII recognize and cleave the following sequences:

In the examples cited above, the EcoRI cleavage created a “cohesive” endwith a 5′ overhang (i.e., the single-stranded “tail” has a 5′ end ratherthan a 3′ end). Cohesive ends can be useful in promoting desiredligations. For example, an EcoRI end is more likely to anneal to anotherEcoRI end than to a HaeIII end.

Over 100 different endonucleases are known, each of which is capable ofcleaving DNA at specific sequences. See, e.g., Roberts, 1982. Allrestriction endonucleases are sensitive to the sequence of bases. Inaddition, some endonucleases are sensitive to whether certain bases havebeen methylated. For example, two endonucleases, MboI and Sau3a arecapable of cleaving the following sequence of bases as shown:

MboI cannot cleave this sequence if the adenine residue is methylated(me-A). Sau3a can cleave this sequence, regardless of whether either Ais methylated. To some extent the methylation (and therefore thecleavage) of a plasmid may be controlled by replicating the plasmids incells with desired methylation capabilities. An E. coli enzyme, DNAadenine methylase (dam), methylates the A residues that occur in GATCsequences. Strains of E. coli which do not contain the dam enzyme aredesignated as dam-cells. Cells which contain dam are designated as dam⁺cells.

Several endonucleases are known which cleave different sequences, butwhich create cohesive ends which are fully compatible with cohesive endscreated by other endonucleases. For example, at least five differentendonucleases create 5′ GATC overhangs, as shown in Table 1.

TABLE 1 Endonuclease Sequence MooI Inhibited by me-A

Sau3a same as MooI Unaffected by me-A BglII Unaffected by me-A

BclI Inhibited by me-A

BamHI Unaffected by me-A

A cohesive end created by any of the endonucleases listed in Table 1will ligate preferentially to a cohesive end created by any of the otherendonucleases. However, a ligation of, for example, a BglII end with aBamHI end will create the following sequence:

This sequence cannot be cleaved by either Bgl II or BamHI; however, itcan be cleaved by MboI (unless methylated) or by Sau3a.

Another endonuclease which involves the GATC sequence is PvuI, whichcreates a 3′ overhang, as follows:

Another endonuclease, ClaI, cleaves the following sequence:

If X₁ is G, or if X₂ is C, then the sequence may be cleaved by MboI(unless methylated, in which case ClaI is also inhibited) or Sau3a.

SUMMARY OF THE INVENTION

This invention relates to chimeric genes which are capable of beingexpressed in plant cells, and to a method for creating such genes.

The chimeric gene comprises a promoter region which is capable ofcausing RNA polymerase in a plant cell to create messenger RNAcorresponding to the DNA. One such promoter region comprises a nopalinesynthase (NOS) promoter region, which normally exists in certain typesof Ti plasmids in bacteria, A. tumefaciens. The NOS gene normally isinactive while contained in A. tumefaciens cells, and it becomes activeafter the Ti plasmid enters a plant cell. Other suitable promoterregions may be derived from genes which exist naturally in plant cells.

The chimeric gene also contains a sequence of bases which codes for a 5′non-translated region of mRNA which is capable of enabling or increasingthe expression in a plant cell of a structural sequence of the mRNA. Forexample, a suitable 5′ non-translated region may be taken from the NOSgene mentioned above, or from a gene which exists naturally in plantcells.

The chimeric gene also contains a desired structural sequence, i.e., asequence which is transcribed into mRNA which is capable of beingtranslated into a desired polypeptide. The structural sequence isheterologous with respect to the promoter region, and it may code forany desired polypeptide, such as a bacterial or mammalian protein. Thestructural sequence includes a start codon and a stop codon. Thestructural sequence may contain introns which are removed from the mRNAprior to translation.

If desired, the chimeric gene may also contain a DNA sequence whichcodes for a 3′ non-translated region (including a poly-adenylationsignal) of mRNA. This region may be derived from a gene which isnaturally expressed in plant cells, to help ensure proper expression ofthe structural sequence. Such genes include the NOS gene mentionedabove, as well as genes which exist naturally in plant cells.

The method of this invention is described below, and is summarized inthe flow chart of FIG. 2.

If properly assembled and inserted into a plant genome, a chimeric geneof this invention will be expressed in the plant cell to create adesired polypeptide, such as a mammalian hormone, or a bacterial enzymewhich confers antibiotic or herbicide resistance upon the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures herein are schematic representations; they have not beendrawn to scale.

FIG. 1 represents the structure of a typical eukaryotic gene.

FIG. 2 is a flow chart representing the steps of this invention,correlated with an example chimeric NOS-NPTII-NOS gene.

FIG. 3 represents fragment HindIII-23, obtained by digesting a Tiplasmid with HindIII.

FIG. 4 represents a DNA fragment which contains a NOS promoter region, aNOS 5′ non-translated region, and the first few codons of the NOSstructural sequence.

FIG. 5 represents the cleavage of a DNA sequence at a precise location,to obtain a DNA fragment which contains a NOS promoter region andcomplete 5′ non-translated region.

FIG. 6 represents the creation of plasmids pMON1001 and pMON40, whichcontain an NPTII structural sequence.

FIG. 7 represents the insertion of a NOS promoter region into plasmidpMON40, to obtain pMON58.

FIG. 8 represents the creation of an M13 derivative designated as M-2,which contains a NOS 3′ non-translated region and poly-A signal.

FIG. 9 represents the assembly of the NOS-NPTII-NOS chimeric gene, andthe insertion of the chimeric gene into plasmid pMON38 to obtainplasmids pMON75 and pMON76.

FIG. 10 represents the insertion of the NOS-NPTII-NOS chimeric gene intoplasmid pMON120 to obtain plasmids pMON128 and pMON129.

FIG. 11 represents the creation of plasmid pMON66, which contains anNPTI gene.

FIG. 12 represents the creation of plasmid pMON73, containing a chimericNOS-NPTII sequence.

FIG. 13 represents the creation of plasmid pMON78, containing a chimericNOS-NPTI sequence.

FIG. 14 represents the creation of plasmids pMON106 and pMON107, whichcontain chimeric NOS-NPTI-NOS genes.

FIG. 15 represents the insertion of a chimeric NOS-NPTI-NOS gene intopMON120 to obtain plasmids pMON130 and pMON131.

FIG. 16 represents the structure of a DNA fragment containing a soybeanprotein (sbss) promoter.

FIG. 17 represents the creation of plasmid pMON121, containing the sbsspromoter.

FIG. 18 represents the insertion of a chimeric sbss-NPTII-NOS gene intopMON120 to create plasmids pMON141 and pMON142.

FIG. 19 represents the creation of plasmid pMON108, containing a bovinegrowth hormone structural sequence and a NOS 3′ region.

FIG. 20 represents the creation of plasmid N25-BGH, which contains theBGH-NOS sequence surrounded by selected cleavage sites.

FIG. 21 represents the insertion of a chimeric sbss-BGH-NOS gene intopMON120 to obtain plasmids pMON147 and pMON148.

FIG. 22 represents the creation of plasmid pMON149, which contains achimeric NOS-BGH-NOS gene.

FIG. 23 represents the creation of plasmid pMON8, which contains astructural sequence for EPSP synthase.

FIG. 24 represents the creation of plasmid pMON25, which contains anEPSP synthase structural sequence with several cleavage sites near thestart codon.

FIG. 25 represents the creation of plasmid pMON146, which contains achimeric sequence comprising EPSP synthase and a NOS 3′ region.

FIG. 26 represents the insertion of a chimeric NOS-EPSP-NOS gene intopMON120 to obtain plasmid pMON153.

FIG. 27 represents the creation of plasmid pMON154, which contains achimeric sbss-EPSP-NOS gene.

DETAILED DESCRIPTION OF THE INVENTION

In one preferred embodiment of this invention, a chimeric gene wascreated which contained the following elements:

1. A promoter region and a 5′ non-translated region derived from anopaline synthase (NOS) gene;

2. A structural sequence derived from a neomycin phosphotransferase II(NPTII) gene; and,

3. A 3′ non-translated region, including a poly-adenylation signal,derived from a NOS gene.

This chimeric gene, referred to herein as a NOS-NPTII-NOS gene, wasassembled and inserted into a variety of plant cells, causing them tobecome resistant to aminoglycoside antibiotics such as kanamycin.

The method used to assemble this chimeric gene is summarized in the flowchart of FIG. 2, and described in detail below and in the examples. Toassist the reader in understanding the steps of this method, variousplasmids and fragments involved in the NOS-NPTII-NOS chimeric gene arecited in parentheses in FIG. 2. However, the method of FIG. 2 isapplicable to a wide variety of other plasmids and fragments. To furtherassist the reader, the steps shown in FIG. 2 have been assigned calloutnumbers 42 et seq. These callout numbers are cited in the followingdescription. The techniques and DNA sequences of this invention arelikely to be useful in the transformation of a wide variety of plants,including any plant which may be infected by one or more strains of A.tumefaciens or A. rhizogenes.

The NOS Promoter Region and 5′ Non-translated Region

The Applicants decided to obtain and utilize a nopaline synthase (NOS)promoter region to control the expression of the heterologous gene. TheNOS is normally carried in certain types of Ti plasmids, such as pTiT37.Sciaky et al., 1978. The NOS promoter is normally inactive while in anA. tumefaciens cell. The entire NOS gene, including the promoter and theprotein coding sequence, is within the T-DNA portion of a Ti plasmidthat is inserted into the chromosomes of plant cells when a plantbecomes infected and forms a crown gall tumor. Once inside the plantcell, the NOS promoter region directs RNA polymerase within a plant cellto transcribe the NOS protein coding sequence into mRNA, which issubsequently translated into the NOS enzyme.

The boundaries between the different parts of a promoter region (shownin FIG. 1 as association region 2, intervening region 4, transcriptioninitiation sequence 6, and intervening region 8), and the boundarybetween the promoter region and the 5′ non-translated region, are notfully understood. The Applicants decided to utilize the entire promoterregion and 5′ non-translated region from the NOS gene, which is known tobe expressed in plant cells. However, it is entirely possible that oneor more of these sequences might be modified in various ways, such asalteration in length or replacement by other sequences. Suchmodifications in promoter regions and 5′ non-translated regions havebeen studied in bacterial cells (see, e.g., Roberts et al 1979) andmammalian cells (see, e.g., McKnight, 1982). By utilizing themethodology taught by this invention, it is now possible to study theeffects of modifications to promoter regions and 5′ non-translatedregions on the expression of genes in plant cells. It may be possible toincrease the expression of a gene in a plant cell by means of suchmodifications. Such modifications, if performed upon chimeric genes ofthis invention, are within the scope of this invention.

A nopaline-type tumor-inducing plasmid, designated as pTiT37, wasisolated from a strain of A. tumefaciens using standard procedures(Currier and Nester, 1976). It was digested with the endonucleaseHindIII which produced numerous fragments. These fragments wereseparated by size on a gel, and one of the fragments was isolated andremoved from the gel. This fragment was designated as the HindIII-23fragment, because it was approximately the 23rd largest fragment fromthe Ti plasmid; it is approximately 3400 base pairs (bp) in size, alsoreferred to as 3.4 kilobases (kb). From work by others (see, e.g.,Hernalsteens et al., 1980), it was known that the HindIII-23 fragmentcontained the entire NOS gene, including the promoter region, a 5′non-translated region, a structural sequence with a start codon and astop codon, and a 3′ non-translated region. The HindIII-23 fragment isshown in FIG. 3.

By means of various cleavage and sequencing experiments, it wasdetermined that the HindIII-23 fragment could be digested by anotherendonuclease, Sau3a, to yield a fragment, about 350 bp in size, whichcontains the entire NOS promoter region, the 5′ non-translated region,and the first few codons of the NOS structural sequence. This fragmentwas sequenced, and the base sequence is represented in FIG. 4. The startcodon (ATG) of the NOS structural sequence begins at base pair 301within the 350 bp fragment. The Applicants decided to cleave thefragment between base pairs 300 and 301; this would provide them with afragment about 300 base pairs long containing a NOS promoter region andthe entire 5′ non-translated region but with no translated bases. Tocleave the 350 bp fragment at precisely the right location, theApplicants obtained an M13 clone designated as S1A, and utilized theprocedure described below.

To create the S1A clone, Dr. Michael Bevan of Washington Universityconverted the 350 bp Sau3a fragment into a single strand of DNA. Thiswas done by utilizing a virus vector, designated as the M13 mp2 phage,which goes through both double-stranded (ds) and single-stranded (ss)stages in its life cycle (Messing et al., lg81). The ds 350 bp fragmentwas inserted into the double-stranded replicative form DNA of the M13mp2, which had been cleaved with BamHI. The two fragments were ligated,and used to infect E. coli cells. The ds DNA containing the 350 bpinserted fragment subsequently replicated, and one strand (the viralstrand) was encapsulated by the M13 viral capsid proteins. In one clone,designated the S1A, the orientation of the 350 bp fragment was such thatthe anti-sense strand (containing the same sequence as the mRNA) of theNOS gene was carried in the viral strand. Viral particles released frominfected cells were isolated, and provided to the Applicants.

Single stranded S1A DNA, containing the anti-sense 350 bp fragment withthe NOS promoter region, was isolated from the viral particles andsequenced. A 14-mer oligonucleotide primer was synthesized, usingpublished procedures (Beaucage and Carruthers, 1981, as modified byAdams et al., 1982). This 14-mer was designed to be complementary tobases 287 through 300 of the 350 bp fragment, as shown on FIG. 4.

The 5′ end of the synthetic primer was radioactively labelled with ³²P;this is represented in the figures by an asterisk.

Copies of the primer were mixed with copies of the single-stranded S1ADNA containing the anti-sense strand of the 350 bp fragment. The primerannealed to the desired region of the S1A DNA, as shown at the top ofFIG. 5. After this occurred, Klenow DNA polymerase and a controlledquantity of unlabelled deoxynucleoside triphosphates (dNTP's), A, T, C,and G, were added. Klenow polymerase added nucleotides to the 3′(unlabelled) end of the primer, but not to the 5′ (labelled) end. Theresult, as shown in FIG. 5, was a circular loop of single-stranded DNA,part of which was matched by a second strand of DNA. The 5′ end of thesecond strand was located opposite base #300 of the Sau3a insert.

The partially double-stranded DNA was then digested by a thirdendonuclease, HaeIII, which can cleave both single-stranded anddouble-stranded DNA. HaeIII cleavage sites were known to exist inseveral locations outside the 350 bp insert, but none existed inside the350 bp insert. This created a fragment having one blunt end, and one 3′overhang which started at base #301 of the Sau3a insert.

The HaeIII fragment mixture was treated with T4 DNA polymerase andunlabelled dNTP's. This caused the single stranded portion of the DNA,which extended from base #301 of the Sau3a insert to the closest HaeIIIcleavage site, to be removed from the fragment. In this manner, the ATGstart codon was removed from base pair #300, leaving a blunt enddouble-stranded fragment which was approximately 550 bp long.

The mixture was then digested by a fourth endonuclease EcoRI, whichcleaved the 550 bp fragment at a single site outside the NOS promoterregion. The fragments were then separated by size on a gel, and theradioactively-labelled fragment was isolated. This fragment containedthe entire NOS promoter region and 5′ non-translated region. It had oneblunt end with a sequence of

and one cohesive end (at the EcoRI site) with a sequence of

The shorter strand was about 308 bp long.

The foregoing steps are represented in FIG. 2 as steps 42, 44, and 46.

This fragment was inserted into pMON40 (which is described below) toobtain pMON58, as shown on FIG. 7.

Creation of Plasmid with NPTII Gene (pMON40)

A bacterial transposon, designated as Tn5, is known to contain acomplete NPTII gene, including promoter region, structural sequence, and3′ non-translated region. The NPTII enzyme inactivates certainaminoglycoside antibiotics, such as kanamycin, neomycin, and G418; seeJimenez and Davies, 1980. This gene is contained within a 1.8 kbfragment, which can be obtained by digesting phage lambda bbkan-1 DNA(D. Berg et al., 1975) with two endonucleases, HindII and BamHI. Thisfragment was inserted into a common laboratory plasmid, pBR327, whichhad been digested by HindIII and BamHI. As shown in FIG. 6, theresulting plasmid was designated as pMON1001, which was about 4.7 kb.

To reduce the size of the DNA fragment which carried the NPTIIstructural sequence, the Applicants eliminated about 500 bp from thepMON1001 plasmid, in the following manner. First, they digested pMON1001at a unique SmaI restriction site which was outside of the NPTII gene.Next, they inserted a 10-mer synthetic oligonucleotide linker,

into the SmaI cleavage site. This eliminated the SmaI cleavage site andreplaced it with a BamHI cleavage site. A second BamHI cleavage sitealready existed, about 500 bp from the new BamHI site. The Applicantsdigested the plasmid with BamHI, separated the 500 bp fragment from the4.2 kb fragment, and circularized the 4.2 kb fragment. The resultingplasmids were inserted into E. coli, which were then selected forresistance to ampicillin and kanamycin. A clonal colony of E. coli wasselected; these cells contained a plasmid which was designated aspMON40, as shown in FIG. 6.

The foregoing steps are represented in FIG. 2 as steps 48 and 50.

Insertion of NOS Promoter into Plasmid pMON40

The Applicants deleted the NPTII promoter from pMON40, and replaced itwith the NOS promoter fragment described previously, by the followingmethod, shown on FIG. 7.

Previous cleavage and sequencing experiments (Rao and Rogers, 1979;Auerswald et al., 1980) indicated that a BglII cleavage site existed inthe NPTII gene between the promoter region and the structural sequence.Plasmid pMON40 was digested with BglII. The cohesive ends were thenfilled in by mixing the cleaved plasmid with Klenow polymerase and thefour dNTP's, to obtain the following blunt ends:

The polymerase and dNTP's were removed, and the cleaved plasmid was thendigested with EcoRI. The smaller fragment which contained the NPTIIpromoter region was removed, leaving a large fragment with one EcoRI endand one blunt end. This large fragment was mixed with the 308 bpfragment which contained the NOS promoter, described previously andshown on FIG. 5. The fragments were ligated, and inserted into E. coli.E coli clones were selected for ampicillin resistance. Replacement ofthe NPTII promoter region (a bacterial promoter) with the NOS promoterregion (which is believed to be active only in plant cells) caused theNPTII structural sequence to become inactive in E. coli. Plasmids from36 kanamycin-sensitive clones were obtained; the plasmid from one clone,designated as pMON58, was utilized in subsequent work.

The foregoing steps are represented in FIG. 2 as steps 52 and 54.

Plasmid pMON58 may be digested to obtain a 1.3 kb EcoRI-BamHI fragmentwhich contains the NOS promoter region, the NOS 5′ non-translatedregion, and the NPTII structural sequence. This step is represented inFIG. 2 as step 56.

Insertion of NOS 3′ Sequence into NPTII Gene

As mentioned above in “Background Art,” the functions of 3′non-translated regions in eucaryotic genes are not fully understood.However, they are believed to contain at least one important sequence, apoly-adenylation signal.

It was suspected by the Applicants that a gene having a bacterial 3′non-translated region might not be expressed as effectively in a plantcell as the same gene having a 3′ non-translated region from a gene,such as NOS, which is known to be expressed in plants. Therefore, theApplicants decided to add a NOS 3 ′ non-translated region to thechimeric gene, in addition to the NPTII 3′ non-translated region alreadypresent. Whether a different type of 3′ non-translated region (such as a3′ region from an octopine-type or agropine-type Ti plasmid, or a 3′region from a gene that normally exists in a plant cell) would besuitable or preferable for use in any particular type of chimeric gene,for use in any specific type of plant cell, may be determined by thoseskilled in the art through routine experimentation using the method ofthis invention. Alternately, it is possible, using the methods describedherein, to delete the NPTII or other existing 3′ non-translated regionand replace it with a desired 3′ non-translated region that is known tobe expressed in plant cells.

Those skilled in the art may also determine through routineexperimentation whether the 3′ non-translated region that naturallyfollows a structural sequence that is to be inserted into a plant cellwill enhance the efficient expression of that structural sequence inthat type of plant cell. If so, then the steps required to insert adifferent 3′ non-translated region into the chimeric gene might not berequired in order to perform the method of this invention.

In order to obtain a DNA fragment containing a NOS 3′ non-translatedregion appropriate for joining to the NPTII structural sequence frompMON58 (described previously), the Applicants utilized a 3.4 kbHindIII-23 fragment from a Ti plasmid, shown on FIG. 3. This 3.4 kbfragment was isolated and digested with BamHI to obtain a 1.1 kbBamHI-HindiIII fragment containing a 3′ portion of the NOS structuralsequence (including the stop codon), and the 3′ non-translated region ofthe NOS gene (including the poly-adenylation signal). This 1.1 kbfragment was inserted into a pBR327 plasmid which had been digested withHindIII and BamHI. The resulting plasmid was designated as pMON42, asshown on FIG. 8.

Plasmid pMON42 was digested with BamHI and RsaI, and a 720 bp fragmentcontaining the desired NOS 3′ non-translated region was purified on agel. The 720 bp fragment was digested with another endonuclease, MboI,and treated with the large fragment of E. coli DNA polymerase I. Thisresulted in a 260 bp fragment with MboI blunt ends, containing a largepart of the NOS 3′ non-translated region including the poly-A signal.

The foregoing procedure is represented in FIG. 2 by step 58. However, itis recognized that alternate means could have been utilized; forexample, it might have been possible to digest the HindIII-23 fragmentdirectly with MboI to obtain the desired 260 bp fragment with the NOS 3′non-translated region.

Assembly of Chimeric Gene

To complete the assembly of the chimeric gene, it was necessary toligate the 260 bp MboI fragment (which contained the NOS 3′non-translated region) to the 1.3 kb EcoRI-BamHI fragment from pMON58(which contained the NOS promoter region and 5′ non-translated regionand the NPTII structural sequence). In order to facilitate this ligationand control the orientation of the fragments, the Applicants decided toconvert the MboI ends of the 260 bp fragment into a BamHI end (at the 5′end of the fragment) and an EcoRI end (at the 3′ end of the fragment).In order to perform this step, the Applicants used the following method.

The 260 bp MboI fragment, the termini of which had been converted toblunt ends by Klenow polymerase, was inserted into M13 mp8 DNA at a SmaIcleavage site. The SmaI site is surrounded by a variety of othercleavage sites present in the M13 mp8 DNA, as shown in FIG. 8. The MboIfragment could be inserted into the blunt SmaI ends in eitherorientation. The orientation of the MboI fragments in different cloneswere tested, using HinfI cleavage sites located asymmetrically withinthe MboI fragment. A clone was selected in which the 3′ end of the NOS3′ non-translated region was located near the EcoRI cleavage site in theM13 mp8 DNA. This clone was designated as the M-2 clone, as shown inFIG. 8.

Replicative form (double stranded) DNA from the M-2 clone was digestedby EcoRI and BamHI and a 280 bp fragment was isolated. Separately,plasmid pMON58 was digested by EcoRI and BamHI, and a 1300 bp fragmentwas isolated. The two fragments were ligated, as shown in FIG. 9, tocomplete the assembly of a NOS-NPTII-NOS chimeric gene having EcoRIends.

There are a variety of ways to control the ligation of the twofragments. For example, the two EcoRI-BamHI fragments could be joinedtogether with DNA ligase and cleaved with EcoRI. After inactivation ofEcoRI, a vector molecule having EcoRI ends that were treated with calfalkaline phosphatase (CAP) may be added to the mixture. The fragments inthe mixture may be ligated in a variety of orientations. The plasmidmixture is used to transform E. coli, and cells having plasmids with thedesired orientation are selected or screened, as described below.

A plasmid, designated as pMON38, was created by insertion of theHindIII-23 fragment (from Ti plasmid pTiT37) into the HindIII cleavagesite of the plasmid pBR327. Plasmid pMON38 contains a unique EcoRI site,and an ampicillin-resistance gene which is expressed in E. coli. PlasmidpMON38 was cleaved with EcoRI and treated with alkaline phosphatase toprevent it from re-ligating to itself. U.S. Pat. No. 4,264,731 (Shine,1981). The resulting fragment was mixed with the 1300 bp NOS-NPTIIfragment from pMON58, and the 280 bp NOS fragment from M-2, which hadbeen ligated and EcoRI-cleaved as described in the previous paragraph.The fragments were ligated, and inserted into E. coli. The E. coli cellswhich had acquired intact plasmids with ampicillin-resistance genes wereselected on plates containing ampicillin. Several clones were selected,and the orientation of the inserted chimeric genes was evaluated bymeans of cleavage experiments. Two clones having plasmids carryingNOS-NPTII-NOS inserts with opposite orientations were selected anddesignated as pMON75 and pMON76, as shown in FIG. 9. The chimeric genemay be isolated by digesting either pMON75 or pMON76 with EcoRI andpurifying a 1580 bp fragment.

The foregoing procedure is represented on FIG. 2 by step 60.

This completes the discussion of the NOS-NPTII-NOS chimeric gene.Additional information on the creation of this gene is provided in theExamples. A copy of this chimeric gene is contained in plasmid pMON128;it may be removed from pMON128 by digestion with EcoRI. A culture of E.coli containing pMON128 has been deposited with the American TypeCulture Collection; this culture -has been assigned accession number39264.

To prove the utility of this chimeric gene, the Applicants inserted itinto plant cells. The NPTII structural sequence was expressed in theplant cells, causing them and their descendants to acquire resistance toconcentrations of kanamycin which are normally toxic to plant cells.

In an alternate preferred embodiment of this invention, a chimeric genewas created comprising (1) a NOS promoter region and 5′ non-translatedregion, (2) a structural sequence which codes for NPT I, and (3) a NOS3′ non-translated region. NPTI and NPTII are different and distinctenzymes with major differences in their amino acid sequences andsubstrate specificities. See, e.g., E. Beck et al., 1982. The relativestabilities and activities of these two enzymes in various types ofplant cells are not yet fully understood, and NPTI may be preferable toNPTII for use in certain types of experiments and plant transformations.

A 1200 bp fragment containing an entire NPTI gene was obtained bydigesting pACYC177 (Chang and Cohen, 1978) with the endonuclease, AvaII.The AvaII termini were converted to blunt ends with Klenow polymerase,and converted to BamHI termini using a synthetic linker. This fragmentwas inserted into a unique BamHI site in a pBR327-derived plasmid, asshown in FIG. 11. The resulting plasmid was designated as pMON66.

Plasmid pMON57 (a deletion derivative of pBR327, as shown in FIG. 11)was digested with AvaII. The 225 bp fragment of pMON57 was replaced bythe analogous 225 bp AvaII fragment taken from plasmid pUC8 (Vieira andMessing, 1982), to obtain a derivative of pMON57 with no PstI cleavagesites. This plasmid was designated as pMON67.

Plasmid pMON58 (described previously and shown in FIG. 7) was digestedwith EcoRI and BamHI to obtain a 1300 bp fragment carrying the NOSpromoter and the NPTII structural sequence.

This fragment was inserted into pMON67 which had been digested withEcoRI and BamHI. The resulting plasmid was designated as pMON73, asshown in FIG. 12.

pMON73 was digested with PstI and BamHI, and a 2.4 kb fragment wasisolated containing a NOS promoter region and 5′ non-translated region.Plasmid pMON66 (shown on FIG. 11) was digested with XhoI and BamHI toyield a 950 bp fragment containing the structural sequence of NPTI. Thisfragment lacked about 30 nucleotides at the 5′ end of the structuralsequence. A synthetic linker containing the missing bases, havingappropriate PstI and XhoI ends, was created. The pMON73 fragment, thepMON66 fragment, and the synthetic linker were ligated together toobtain plasmid pMON78, as shown in FIG. 13. This plasmid contains theNOS promoter region and 5′ non-translated region adjoined to the NPTIstructural sequence. The ATG start codon was in the same position thatthe ATG start codon of the NOS structural sequence had occupied.

Plasmid pMON78 was digested with EcoRI and BamHI to yield a 1300 bpfragment carrying the chimeric NOS-NPTI regions. Double-stranded DNAfrom the M-2 clone (described previously and shown on FIG. 9) wasdigested with EcoRI and BamHI, to yield a 280 bp fragment carrying a NOS3′ non-translated region with a poly-adenylation signal. The twofragments described above were ligated together to create theNOS-NPTI-NOS chimeric gene, which was inserted into plasmid pMON38(described above) which had been digested with EcoRI. The two resultingplasmids, having chimeric gene inserts with opposite orientations, weredesignated as pMON106 and pMON107, as shown in FIG. 14.

Either of plasmids pMON106 or pMON107 may be digested with EcoRI toyield a 1.6 kb fragment containing the chimeric NOS-NPTI-NOS gene. Thisfragment was inserted into plasmid pMON120 which had been digested withEcoRI and treated with alkaline phosphatase. The resulting plasmids,having inserts with opposite orientations, were designated as pMON130and pMON131, as shown on FIG. 15.

The NOS-NPTI-NOS chimeric gene was inserted into plant cells, whichacquired resistance to kanamycin. This demonstrates expression of thechimeric gene in plant cells.

Creation of Chimeric Gene with Soybean Promoter

In an alternate preferred embodiment of this invention, a chimeric genewas created comprising (1) a promoter region and 5′ non-translatedregion taken from a gene which naturally exists in soybean; this genecodes for the small subunit of ribulose-1,5-bisphosphate carboxylase(sbss for soybean small subunit); (2) a structural sequence which codesfor NPTII, and (3) a NOS 3′ non-translated region.

The sbss gene codes for a protein in soybean leaves which is involved inphotosynthetic carbon fixation. The sbss protein is the most abundantprotein in soybean leaves (accounting for about 10% of the total leafprotein), so it is likely that the sbss promoter region causes prolifictranscription.

There are believed to be approximately six genes encoding the ssRuBPCase protein in the soybean genome. One of the members of the ssRuBPCase gene family, SRS1, which is highly transcribed in soybeanleaves, has been cloned and characterized. The promoter region, 5′non-translated region, and a portion of the structural sequence arecontained on a 2.1 kb EcoRI fragment that was subcloned into the EcoRIsite of plasmid pBR325 (Bolivar, 1978). The resultant plasmid, pSRS2.1,was a gift to Monsanto Company from Dr. R. B. Meagher, University ofGeorgia, Athens, Ga. The 2.1 kb EcoRI fragment from pSRS2.1 is shown onFIG. 16.

Plasmid pSRS2.1 was prepared from dam⁻ E. coli cells, and cleaved withMboI to obtain an 800 bp fragment. This fragment was inserted intoplasmid pKC7 (Rao and Rogers, 1979) which had been cleaved with BglII.The resulting plasmid was designated as pMON121, as shown on FIG. 17.

Plasmid pMON121 was digested with EcoRI and BclI, and a 1200 bp fragmentcontaining the sbss promoter region was isolated. Separately, plasmidpMON75 (described previously and shown on FIG. 9) was digested withEcoRI and BglII, and a 1250 bp fragment was isolated, containing a NPTIIstructural sequence and a NOS 3′ non-translated region. The twofragments were ligated at the compatible BclI/BglII overhangs, to createa 2450 bp fragment containing sbss-NPTII-NOS chimeric gene. Thisfragment was inserted into pMON120 which had been cleaved with EcoRI, tocreate two plasmids having chimeric gene inserts with oppositeorientations, as shown in FIG. 18. The plasmids were designated aspMON141 and pMON142.

The sbss-NPTII-NOS chimeric genes were inserted into several types ofplant cells, causing the plant cells to acquire resistance to kanamycin.

This successful transformation proved that a promoter region from onetype of plant can cause the expression of a gene within plant cells froman entirely different genus, family, and order of plants.

The chimeric sbss-NPTII-NOS gene also had another significant feature.Sequencing experiments indicated that the 800 bp MboI fragment containedthe ATG start codon of the sbss structural sequence. Rather than removethis start codon, the Applicants decided to insert a stop codon behindit in the same reading frame.

This created a dicistronic mRNA sequence, which coded for a truncatedamino portion of the sbss polypeptide and a complete NPTII polypeptide.Expression of the NPTII polypeptide was the-first proof that adicistronic mRNA can be translated within plant cells.

The sbss promoter is contained in plasmid pMON154, described below. Aculture of E. coli containing this plasmid has been deposited with theAmerican Type Culture Center. This culture has been assigned accessionnumber 39265.

Creation of BGH Chimeric Genes

In an alternate preferred embodiment of this invention, a chimeric genewas created comprising (1) a sbss promoter region and 5′ non-translatedregion, (2) a structural sequence which codes for bovine growth hormone(BGH) and (3) a NOS 3′ non-translated region. This chimeric gene wascreated as follows.

A structural sequence which codes for the polypeptide, bovine growthhormone, (see, e.g., Woychik et al., 1982) was inserted into apBR322-derived plasmid. The resulting plasmid was designated as plasmidCP-1. This plasmid was digested with EcoRI and HindIII to yield a 570 bpfragment containing the structural sequence. Double stranded M-2 RF DNA(described previously and shown in FIG. 8) was cleaved with EcoRI andHindIII to yield a 290 bp fragment which contained the NOS 3′non-translated region with a poly-adenylation signal. The two fragmentswere ligated together and digested with EcoRI to create an 860 base pairfragment with EcoRI ends, which contained a BGH-coding structuralsequence joined to the NOS 3′ non-translated region. This fragment wasintroduced into plasmid pMON38, which had been digested with EcoRI andtreated with alkaline phosphatase, to create a new plasmid, designatedas pMON108, as shown in FIG. 19.

A unique BglII restriction site was introduced at the 5′ end of the BGHstructural sequence by digesting PMON 108 with EcoRI to obtain the 860bp fragment, and using Klenow polymerase to create blunt ends on theresulting EcoRI fragment. This fragment was ligated into plasmid N25 (aderivative of pBR327 containing a synthetic linker carrying BglII andXbaI cleavage sites inserted at the BamHI site), which had been cleavedwith XbaI and treated with Klenow polymerase to obtain blunt ends (N25contains a unique BglII site located 12 bases from the XbaI site). Theresulting plasmid, which contained the 860 bp BGH-NOS fragment in theorientation shown in FIG. 20, was designated as plasmid N25-BGH. Thisplasmid contains a unique BglII cleavage site located about 25 basesfrom the 5′ end of the BGH structural sequence.

Plasmid N25-BGH prepared from dam⁻ E. coli cells was digested with BglIIand ClaI to yield an 860 bp fragment which contained the BGH structuralsequence joined to the NOS 3′ non-translated region. Separately, plasmidpMON121 (described previously and shown in FIG. 17) was prepared fromdam⁻ E. coli cells and was digested with ClaI and BclI to create an 1100bp fragment which contained the sbss promoter region. The fragments wereligated at their compatible BclI/BglII overhangs, and digested with ClaIto yield a ClaI fragment of about 2 kb containing the chimericsbss-BGH-NOS gene. This fragment was inserted into pMON120 (describedpreviously and shown in FIG. 10) which had been digested with ClaI. Theresulting plasmids, containing the inserted chimeric gene in oppositeorientations were designated pMON147 and pMON148, as shown in FIG. 21.

An alternate chimeric BGH gene was created which contained (1) a NOSpromoter region and 5′ non-translated region, (2) a structural sequencewhich codes for BGH, and (3) a NOS 3′ non-translated region, by thefollowing method, shown in FIG. 22.

Plasmid pMON76 (described above and shown in FIG. 9) was digested withEcoRI and BglII to obtain a 308 bp fragment containing a NOS promoterregion and 5′ non-translated region. Plasmid N25-BGH prepared from dam⁻E. coli cells (described above and shown in FIG. 20) was digested withBglII and ClaI to obtain a 900 bp fragment containing a BGH structuralsequence and a NOS 3′ non-translated region. These two fragments wereligated together to obtain a chimeric NOS-BGH-NOS gene in a fragmentwith EcoRI and ClaI ends. This fragment was ligated with an 8 kbfragment obtained by digesting pMON120 with EcoRI and Cla. The resultingplasmid, designated as pMON149, is shown in FIG. 22.

Creation of Chimeric NOS-EPSP-NOS Gene

In an alternate preferred embodiment, a chimeric gene was createdcomprising (1) a NOS promoter region and 5′ non-translated region, (2) astructural sequence which codes for the E. coli enzyme,5-enolpyruvylshikimate-3-phosphoric acid synthase (EPSP synthase) and(3) a NOS 3′ non-translated region.

EPSP synthase is believed to be the target enzyme for the herbicide,glyphosate, which is marketed by Monsanto Company under the registeredtrademark, “Roundup.” Glyphosate is known to inhibit EPSP synthaseactivity (Amrhein et al., 1980), and amplification of the EPSP synthasegene in bacteria is known to increase their resistance to glyphosate.Therefore, increasing the level of EPSP synthase activity in plants mayconfer resistance to glyphosate in transformed plants. Since glyphosateis toxic to most plants, this provides for a usefull method of weedcontrol. Seeds of a desired crop plant which has been transformed toincrease EPSP synthase activity may be planted in a field. Glyphosatemay be applied to the field at concentrations which will kill allnon-transformed plants, leaving the non-transformed plants unharmed.

An EPSP synthase gene may be isolated by a variety of means, includingthe following. A lambda phage library may be created which carries avariety of DNA fragments produced by HindIII cleavage of E. coli DNA.See, e.g., Maniatis et al., 1982.

The EPSP synthase gene is one of the genes which are involved in theproduction of aromatic amino acids. These genes are designated as the“aro” genes; EPSP synthase is designated as aroA. Cells which do notcontain functional aro genes are designated as aro⁻ cells. Aro⁻ cellsmust normally be grown on media supplemented by aromatic amino acids.See Pittard and Wallis, 1966.

Different lambda phages which carry various HindIII fragments may beused to infect mutant E. coli cells which do not have EPSP synthasegenes. The infected aro⁻ cells may be cultured on media which does notcontain the aromatic amino acids, and transformed aro⁺ clones which arecapable of growing on such media may be selected. Such clones are likelyto contain the EPSP synthase gene. Phage particles may be isolated fromsuch clones, and DNA may be isolated from these phages. The phage DNAmay be cleaved with one or more restriction endonucleases, and by agradual process of analysis, a fragment which contains the EPSP synthasegene may be isolated.

Using a procedure similar to the method summarized above, the Applicantsisolated an 11 kb HindIII fragment which contained the entire E. coliEPSP synthase gene. This fragment was digested with BglII to produce a3.5 kb HindIII-BglII fragment which contained the entire EPSP synthasegene. This 3.5 kb fragment was inserted into plasmid pKC7 (Rao andRogers, 1979) to produce plasmid pMON4, which is shown in FIG. 23.

Plasmid pMON4 was digested with ClaI to yield a 2.5 kb fragment whichcontained the EPSP synthase structural sequence. This fragment wasinserted into pBR327 that had been digested with ClaI, to create pMON8,as shown in FIG. 23.

pMON8 was digested with BamHI and NdeI to obtain a 4.9 kb fragment. Thisfragment lacked about 200 nucleotides encoding the amino terminus of theEPSP synthase structural sequence.

The missing nucleotides were replaced by ligating a HinfI/NdeI fragment,obtained from pMON8 as shown in FIG. 24, together with a syntheticoligonucleotide sequence containing (1) the EPSP synthase start codonand the first three nucleotides, (2) a unique BglII site, and (3) theappropriate BamHI and HinfI ends. The resulting plasmid, pMON25,contains an intact EPSP synthase structural sequence with unique BamHIand BglII sites positioned near the start codon.

Double stranded M-2 DNA (described previously and shown in FIG. 8) wasdigested with HindIII and EcoRI to yield a 290 bp fragment whichcontains the NOS 3′ non-translated region and poly-adenylation signal.This fragment was introduced into a pMON25 plasmid that had beendigested with EcoRI and HindIII to create a plasmid, designated aspMON146 (shown in FIG. 25) which contains the EPSP structural sequencejoined to the NOS 3′ non-translated region.

pMON146 was cleaved with ClaI and BglII to yield a 2.3 kb fragmentcarrying the EPSP structural sequence joined to the NOS 3′non-translated region. pMON76 (described previously and shown in FIG. 9)was digested with BglII and EcoRI to create a 310 bp fragment containingthe NOS promoter region and 5′ non-translated region. The abovefragments were mixed with pMON120 (described previously and shown inFIG. 10) that had been digested with ClaI and EcoRI, and the mixture wasligated. The resulting plasmid, designated pMON153, is shown in FIG. 26.This plasmid contains the chimeric NOS-EPSP-NOS gene.

A plasmid containing a chimeric sbss-EPSP-NOS gene was prepared in thefollowing manner, shown in FIG. 27. Plasmid pMON146 (describedpreviously and shown in FIG. 25) was digested with ClaI and BglII, and a2.3 kb fragment was purified. This fragment contained the EPSP synthasestructural sequence coupled to a NOS 3′ non-translated region with apoly-adenylation signal. Plasmid pMON121 (described above and shown inFIG. 17) was digested with ClaI and BclI, and a 1.1 kb fragment waspurified. This fragment contains an sbss promoter region and 5′non-translated region. The two fragments were mixed and ligated with T4DNA ligase and subsequently digested with ClaI. This created a chimericsbss-EPSP-NOS gene, joined through compatible BglII and BclI termini.This chimeric gene with ClaI termini was inserted into plasmid pMON120which had been digested with ClaI and treated with calf alkalinephosphatase (CAP). The mixture was ligated with T4 DNA ligase. Theresulting mixture of fragments and plasmids was used to transform E.coli cells, which were selected for resistance to spectinomycin. Acolony of resistant cells was isolated, and the plasmid in this colonywas designated as pMON154, as shown in FIG. 27.

A culture of E. coli containing pMON154 has been deposited with theAmerican Type Culture Center. This culture has been assigned accessionnumber 39265.

Means for Inserting Chimeric Genes into Plant Cells

A variety of methods are known for inserting foreign DNA into plantcells. One such method, utilized by the Applicants, involved inserting achimeric gene into Ti plasmids carried by A. tumefaciens, andco-cultivating the A. tumefaciens cells with plants. A segment of T-DNAcarrying the chimeric gene was transferred into the plant genome,causing transformation. This method is described in detail in twoseparate, simultaneously-filed in two separate, simultaneously filedapplications entitled “Plasmids for Transforming Plant Cells,” MonsantoCase Number C-07-21-(133) U.S. Ser. No. 458,411, filed Jan. 17, 1983 nowabandoned, and “Genetically Transformed Plants,” Monsanto Case NumberC-07-21-(134) U.S. Ser. No. 458,402, filed Jan. 17, 1983 now abandoned.The contents of both of those applications are hereby incorporated byreference.

A variety of other methods are listed below. These methods aretheoretically capable of inserting the chimeric genes of this inventioninto plant cells, although the reported transformation efficienciesachieved to date by such methods have been low. The chimeric genes ofthis invention (especially those chimeric genes such as NPTI and NPTII,which may be utilized as selectable markers) are likely to facilitateresearch on methods of inserting DNA into plants or plant cells.

1. One alternate technique for inserting DNA into plant cells involvesthe use of lipid vesicles, also called liposomes. Liposomes may beutilized to encapsulate one or more DNA molecules. The liposomes andtheir DNA contents may be taken up by plant cells; see, e.g., Lurquin,1981. If the inserted DNA can be incorporated into the plant genome,replicated, and inherited, the plant cells will be transformed.

To date, efforts to use liposomes to deliver DNA into plant cells havenot met with great success (Fraley and Papahadjopoulos, 1981). Onlyrelatively small DNA molecules have been transferred into plant cells bymeans of liposomes, and none have yet been expressed. However,liposome-delivery technology is still being actively developed, and itis likely that methods will be developed for transferring plasmidscontaining the chimeric genes of this invention into plant cells bymeans involving liposomes.

2. Other alternate techniques involve contacting plant cells with DNAwhich is complexed with either (a) polycationic substances, such aspoly-L-ornithine (Davey et al., 1980), or (b) calcium phosphate (Krenset al., 1982). Although efficiencies of transformation achieved to datehave been low, these methods are still being actively researched.

3. A method has been developed involving the fusion of bacteria, whichcontain desired plasmids, with plant cells. Such methods involveconverting the bacteria into spheroplasts and converting the plant cellsinto protoplasts. Both of these methods remove the cell wall barrierfrom the bacterial and plant cells, using enzymic digestion. The twocell types can then be fused together by exposure to chemical agents,such as polyethylene glycol. See Hasezawa et al., 1981. Although thetransformation efficiencies achieved to date by this method have beenlow, similar experiments using fusions of bacterial and animal cellshave produced good results; see Rassoulzadegan et al., 1982.

4. Two other methods which have been used successfully to geneticallytransform animal cells involve (a) direct microinjection of DNA intoanimal cells, using very small glass needles (Capecchi, 1980), and (b)electric-current-induced uptake of DNA by animal cells (Wong andNeumann, 1982). Although neither of these techniques have been utilizedto date to transform plant cells, they may be useful to insert numericgenes of this invention into plant cells.

Use of Chimeric Genes to Identify Plant Regulators

The chimeric genes of this invention may be used to identify, isolate,and study DNA sequences to determine whether they are capable ofpromoting or otherwise regulating the expression of genes within plantcells.

For example, the DNA from any type of cell can be fragmented, usingpartial endonuclease digestion or other methods. The DNA fragments aremixed with multiple copies of a chimeric gene which has been cleaved ata unique cleavage site that is located in the 5′ direction from the ATGstart codon of the structural sequence. Preferably, the structuralsequence, if properly transcribed, will be translated into a selectablemarker, such as a polypeptide which confers resistance on the host to aselected antibiotic. The DNA mixture is ligated to form plasmids, andthe plasmids are used to transform plant cells which are sensitive tothe selected antibiotic. The cells are cultured on media which containsan appropriate concentration of the selected antibiotic. Plant cellswill survive and reproduce only if the structural sequence istranscribed and translated into the polypeptide which confers resistanceto the antibiotic. This is presumed to occur only if the inserted DNAfragment performs the function of a gene promoter; the resistantcolonies will be evaluated further to determine whether this is thecase.

Using this technique, it is possible to evaluate the promoter regions ofbacteria, yeast, fungus, algae, other microorganisms, and animal cells,to determine whether they also function as gene promoters in varioustypes of plant cells. It is also possible to evaluate promoters from onetype of plant in other types of plant cells. By using similar methodsand varying the cleavage site in the chimeric gene, it is possible toevaluate the performance of any DNA sequence as a 5′ non-translatedregion, a 3′ non-translated region, or any type of other regulatorysequence.

If desired, a partial chimeric gene may be utilized in this method ofevaluating the regulatory effects of various DNA sequences. For example,the NOS promoter region and/or the NOS 5′ non-translated region may bedeleted from the NOS-NPTII-NOS chimeric gene. This would create achimeric gene having a unique cleavage site but no promoter region infront of an NPTII structural sequence.

In case the inserted DNA fragment contains a start codon which might (1)alter the reading frame of the structural sequence, or (2) alter theamino terminus of the polypeptide, it is possible to place anoligonucleotide between the cleavage site and the start codon of thestructural sequence. The oligonucleotide would contain stop codons inall three reading frames. Therefore, if a start codon was included inthe inserted DNA fragment, the gene would be a dicistronic gene. Thefirst polypeptide would be terminated by whichever stop codon happenedto be in the reading frame of the inserted start codon. The second startcodon would begin the translation of a separate polypeptide, which wouldbe the selectable marker enzyme.

Meaning of Various Phrases

A variety of phrases which are used in the claims must be defined anddescribed to clarify the meaning and coverage of the claims.

The meaning of any particular term shall be interpreted with referenceto the text and figures of this application. In particular, it isrecognized that a variety of terms have developed which are usedinconsistently in the literature. For example, a variety of meaningshave evolved for the term “promoter,” some of which include the 5′non-translated region and some of which do not. In an effort to avoidproblems of interpretation, the Applicants have attempted to definevarious terms. However, such definitions are not presumed or intended tobe comprehensive and they shall be interpreted in light of the relevantliterature.

The term “chimeric gene” refers to a gene that contains at least twoportions that were derived from different and distinct genes. As usedherein, this term is limited to genes which have been assembled,synthesized, or-otherwise produced as a result of man-made efforts, andany genes which are replicated or otherwise derived therefrom. “Man-madeefforts” include enzymatic, cellular, and other biological processes, ifsuch processes occur under conditions which are caused, enhanced, orcontrolled by human effort or intervention; this excludes genes whichare created solely by natural processes.

As used herein, a “gene” is limited to a segment of DNA which isnormally regarded as a gene by those skilled in the art. For example, aplasmid might contain a plant-derived promoter region and a heterologousstructural sequence, but unless those two segments are positioned withrespect to each other in the plasmid such that the promoter regioncauses the transcription of the structural sequence, then those twosegments would not be regarded as included in the same gene.

This invention relates to chimeric genes which have structural sequencesthat are “heterologous” with respect to their promoter regions. Thisincludes at least two types of chimeric genes:

1. DNA of a gene which is foreign to a plant cell. For example, if astructural sequence which codes for mammalian protein or bacterialprotein is coupled to a plant promoter region, such a gene would beregarded as heterologous.

2. A plant cell gene which is naturally promoted by a different plantpromoter region. For example, if a structural sequence which codes for aplant protein is normally controlled by a low-quantity promoter, thestructural sequence may be coupled with a prolific promoter. This mightcause a higher quantity of transcription of the structural sequence,thereby leading to plants with higher protein content. Such a structuralsequence would be regarded as heterologous with regard to the prolificpromoter.

However, it is not essential for this invention that the entirestructural sequence be heterologous with respect to the entire promoterregion. For example, a chimeric gene of this invention may be createdwhich would be translated into a “fusion protein,” i.e., a proteincomprising polypeptide portions derived from two separate structuralsequences. This may be accomplished by inserting all or part of aheterologous structural sequence into the structural sequence of a plantgene, somewhere after the start codon of the plant structural sequence.

As used herein, the phrase, “a promoter region derived from a specifiedgene” shall include a promoter region if one or more parts of thepromoter region were derived from the specified gene. For example, itmight be discovered that one or more portions of a particularplant-derived promoter region (such as intervening region 8, shown onFIG. 1) might be replaced by one or more sequences derived from adifferent gene, such as the gene that contains the heterologousstructural sequence, without reducing the expression of the resultingchimeric gene in a particular type of host cell. Such a chimeric genewould contain a plant-derived association region 2, intervening region4, and transcription initiation sequence 6, followed by heterologousintervening region 8, 5′ non-translated region 10 and structuralsequence 14. Such a chimeric gene is within the scope of this invention.

As used herein, the phrase “derived from” shall be construed broadly.For example, a structural sequence may be “derived from” a particulargene by a variety of processes, including the following:

1. The gene may be reproduced by various means such as inserting it intoa plasmid and replicating the plasmid by cell culturing, in vitroreplication, or other methods, and the desired sequence may be obtainedfrom the DNA copies by various means such as endonuclease digestion;

2. mRNA which was coded for by the gene may be obtained and processed invarious ways, such as preparing complementary DNA from the mRNA and thendigesting the cDNA with endonucleases;

3. The sequence of bases in the structural sequence may be determined byvarious methods, such as endonuclease mapping or the Maxam-Gilbertmethod. A strand of DNA which duplicates or approximates the desiredsequence may be created by various methods, such as chemical synthesisor ligation of oligonucleotide fragments.

4. A structural sequence of bases may be deduced by applying the geneticcode to the sequence of amino acid residues in a polypeptide.

Usually, a variety of DNA structural sequences may be determined for anypolypeptide, because of the redundancy of the genetic code. From thisvariety, a desired sequence of bases may be selected, and a strand ofDNA having the selected sequence may be created.

If desired, any DNA sequence may be modified by substituting certainbases for the existing bases. Such modifications may be performed for avariety of reasons. For example, one or more bases in a sequence may bereplaced by other bases in order to create or delete a cleavage site fora particular endonuclease. As another example, one or more bases in asequence may be replaced in order to reduce the occurrence of “stem andloop” structures in messenger RNA. Such modified sequences are withinthe scope of this invention.

A structural sequence may contain introns and exons; such a structuralsequence may be derived from DNA, or from an mRNA primary transcript.Alternately, a structural sequence may be derived from processed mRNA,from which one or more introns have been deleted.

The Applicants have deposited two cultures of E. coli cells containingplasmids pMON128 and pMON154 with the American Type Culture Collection(ATCC). These cells have been assigned ATCC accession numbers 39264 and39265, respectively. The Applicants have claimed cultures ofmicroorganisms having the “relevant characteristics” of either culture.As used herein, the “relevant characteristics” of a cell culture arelimited to those characteristics which make the culture suitable for ause which is disclosed, suggested or made possible by the informationcontained herein. Numerous characteristics of the culture may bemodified by techniques known to those skilled in the art; for example,the cells may be made resistant to a particular antibiotic by insertionof a particular plasmid or gene into the cells, or the pMON128 orpMON154 plasmids might be removed from the designated cells and insertedinto a different strain of cells. Such variations are within the scopeof this invention, even though they may amount to improvements, whichundoubtedly will occur after more researchers gain access to these cellcultures.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific embodiments described herein. Such equivalents are within thescope of this invention.

EXAMPLES Example 1 Creation of pMON1001

Fifty micrograms (ug) of lambda phage bbkan-1 DNA (Berg et al., 1975)were digested with 100 units of HindIII (all restriction endonucleaseswere obtained from New England Biolabs, Beverly, Mass., and were usedwith buffers according to the suppliers instructions, unless otherwisespecified) for 2 hours at 37° C. After heat-inactivation (70° C., 10minutes), the 3.3 kb Tn5 HindIII fragment was purified on gradient. Oneug of the purified HindIII fragment was digested with BamHI (2 units, 1hour, 37° C.), to create a 1.8 kb fragment. The endonuclease was heatinactivated.

Plasmid pBR327 (Soberon et al., 1981), 1 ug, was digested with HindIIIand BamHI (2 units each, 2 hours, 37° C.). Following digestion, theendonucleases were heat inactivated and the cleaved pBR327 DNA was addedto the BamHI-HindIII Tn5 fragments. After addition of ATP to aconcentration of 0.75 mM, 10 units of T4 DNA ligase (prepared by themethod of Murray et al., 1979) was added, and the reaction was allowedto continue for 16 hours at 12-14° C. One unit of T4 DNA ligase willgive 90% circularization of one ug of HindIII-cleaved pBR327 plasmid in5 minutes at 22° C.

The ligated DNA was used to transform CaCl₂-shocked E. coli C600 recA56cells (Maniatis et al., 1982). After expression in Luria broth (LB) for1 hour at 37° the cells were spread on solid LB media plates containing200 ug/ml ampicillin and 40 ug/ml kanamycin. Following 16 hoursincubation at 37° C., several hundred colonies appeared. Plasmidmini-prep DNA was prepared from six of these. (Ish-Horowicz and Burke,1981). Endonuclease digestion showed that all six of the plasmidscarried the 1.8 kb HindIII-BamHI fragment. One of those isolates wasdesignated as pMON1001 as shown in FIG. 6.

Example 2 Creation of pMON40

Five ug of plasmid pMON1001 (described in Example 1) was digested withSmaI. The reaction was terminated by phenol extraction, and the DNA wasprecipitated by ethanol. A BamHI linker CCGGATCCGG (0.1 ug), which hadbeen phosphorylated with ATP and T4 polynucleotide kinase (BethesdaResearch Laboratory, Rockville, Md.) was added to 1 ug of the pMON1001fragment. The mixture was treated with T4 DNA ligase (100 units) for 18hours at 14° C. After heating at 70° C. for 10 minutes to inactivate theDNA ligase, the DNA mixture was digested with BamHI endonuclease (20units, 3 hours, 37° C.) and separated by electrophoresis on an 0.5%agarose gel. The band corresponding to the 4.2 kb SmaI-BamHI vectorfragment was excised from the gel. The 4.2 kb fragment was purified byabsorption on glass beads (Vogelstein and Gillespie, 1979), ethanolprecipitated and resuspended in 20 ul of DNA ligase buffer with ATP. T4DNA ligase (20 units) was added and the mixture was incubated for 1.5hours at room temperature. The DNA was mixed with rubidiumchloride-shocked in E. coli C600 cells for DNA transformation. (Maniatiset al., 1982). After expression for 1 hour at 37° C. in LB, the cellswere spread on LB plates containing 200 ug/ml of ampicillin and 20 ug/mlkanamycin. The plates were incubated at 37° C. for 16 hours. Twelveampicillin-resistant, kanamycin-resistant colonies were chosen, 2 mlcultures were grown, and mini-plasmid preparations were performed.Endonuclease mapping of the plasmids revealed that ten of the twelvecontained no SmaI site and a single BamHi site, and were of theappropriate size, 4.2 kb. The plasmid from one of the ten colonies wasdesignated as pMON40, as shown in FIG. 6.

Example 3 Creation of NOS Promoter Fragment

An oligonucleotide with the following sequence, 5′-TGCAGATTATTTGG-3′,was synthesized (Beaucage and Carruthers, 1981, as modified by Adams etal., 1982). This oligonucleotide contained a ³²P radioactive label,which was added to the 5′ thymidine residue by polynucleotide kinase.

An M13 mp7 derivative, designated as S1A, was given to Applicants by M.Bevan and M. D. Chilton, Washington University, St. Louis, Mo. To thebest of Applicants' knowledge and belief, the S1A DNA was obtained bythe following method. A pTiT37 plasmid was digested with HindIII, and a3.4 kb fragment was isolated and designated as the HindIII-23 fragment.This fragment was digested with Sau3a, to create a 344 bp fragment withSau3a ends. This fragment was inserted into double-stranded, replicativeform DNA from the M13 mp7 phage vector (Messing et al., 1981) which hadbeen cut with BamHI. Two recombinant phages with 344 bp insertsresulted, one of which contained the anti-sense strand of the NOSpromoter fragment. That recombinant phage was designated as S1A, and aclonal copy was given to the Applicants.

The Applicants prepared the single-stranded form of the S1A DNA (14. 4ug, 6 pmol), and annealed it (10 minutes at 70° C., then cooled to roomtemperature) with 20 pmol of the 14-mer oligonucleotide, mentionedabove. The oligonucleotide annealed to the Sau3a insert at bases 286-300as shown on FIGS. 4 and 5.

200 ul of the S1A template and annealed oligonucleotide were mixed withthe four dNTP's (present at a final concentration of lmM, 25 ul) and 50ul of Klenow polymerase. The mixture incubated for 30 minutes at roomtemperature. During this period, the polymerase added dNTP's to the 3′end of the oligonucleotide. The polymerase was heat-inactivated (70° C.,3 minutes), and HaeIII (160 units) were added. The mixture was incubated(1 hour, 55° C.), the HaeIII was inactivated (70° C., 3 minutes), andthe four dNTP's (1 mM, 12 ul) and T4 DNA polymerase (50 units) wereadded. The mixture was incubated (1 hour, 37° C.) and the polymerase wasinactivated (70° C., 3 minutes). This yielded a fragment of about 570bp. EcoRI (150 units) was added, the mixture was incubated (1 hour, 37°C.) and the EcoRI was inactivated (70° C., 3 minutes).

Aliquots of the mixture were separated on 6% acrylamide with 25%glycerol. Autoradiography revealed a radioactively labelled band about310 bp in size. This band was excised. The foregoing procedure isindicated by FIG. 5.

Example 4 Creation of pMON58

Five ug of plasmid pMON40 (described in Example 2) were digested withBglII (10 units, 1.5 hours, 37° C.), and the BglII was inactivated (70°C., 10 minutes). The four dNTP's (1 mM, 5 ul) and Klenow polymerase (8units) were added, the mixture was incubated (37° C., 40 minutes), andthe polymerase was inactivated (70° C., 10 minutes). EcoRI (10 units)was added and incubated (1 hour, 37° C.), and calf alkaline phosphatase(CAP) was added and incubated (1 hour, 37° C.). A fragment of about 3.9kb was purified on agarose gel using NA-45 membrane (Scheicher andScheull, Keene NH). The fragment (1.0 pM) was mixed with the NOSpromoter fragment (0.1 pM), described in Example 3, and with T4 DNAligase (100 units). The mixture was incubated (4° C., 16 hours). Theresulting plasmids were inserted into E. coli cells, which were selectedon media containing 200 ug/ml ampicillin. Thirty-six clonal Amp^(R)colonies were selected, and mini-preps of plasmids were made from thosecolonies. The plasmid from one colony demonstrated a 308 bp EcoRI-BglIIfragment, a new SstII cleavage site carried by the 308 bp NOS fragment,and a new PstI site. This plasmid was designated as pMON58, as shown inFIG. 7. pMON58 DNA was prepared as described above.

Example 5 Creation of pMON42

Plasmid pBR325-HindIII-23, a derivative of plasmid pBR325 (Bolivar,1978) carrying the HindIII-23 fragment of pTIT37 (see FIG. 3) in theHindIII site, was given to Applicants by M. Bevan and M. D. Chilton,Washington University, St. Louis, Mo. DNA of this plasmid was preparedand 30 ug were digested with HindIII (50 units) and BamHI (50 units).The 1.1 kb HindIII-BamHI fragment was purified by adsorption on glassbeads (Vogelstein and Gillespie, 1979) after agarose gelelectrophoresis. The purified fragment (0.5 ug) was added to 0.5 ug ofthe 2.9 kb HindIII-BamHI fragment of pBR327. After treatment with DNAligase (20 units, 4 hours, 22° C.), the resulting plasmids wereintroduced to E. coli C600 cells. Clones resistant to ampicillin at 200ug/ml were selected on solid media; 220 clones were obtained. Miniprepsof plasmid DNA were made from six of these clones and tested with thepresence of a 1.1 kb fragment after digestion with HindIII and BamHI.One plasmid which demonstrated the correct insert was designated pMON42.Plasmid pMON42 DNA was prepared as described in previous examples.

Example 6 Creation of M13 Clone M-2

Seventy-five ug of plasmid pMON42 (described in Example 5) prepared fromdam⁻ E. coli cells were digested with RsaI and BamHI (50 units of each,3 hours, 37° C.) and the 720 bp RsaI-BamHI fragment was purified usingNA-45 membrane. Eight ug of the purified 720 bp BamHI-RsaI fragment weredigested with MboI (10 minutes, 70° C.), the ends were made blunt byfilling in with the large Klenow fragment of DNA polymerase I and thefour dNTP's. Then 0.1 ug of the resulting DNA mixture was added to 0.05ug of M13 mp8 previously digested with SmaI (1 unit, 1 hour 37° C.) andcalf alkaline phosphatase (0.2 units). After ligation (10 units of T4DNA ligase, 16 hours, 12° C.) and transfection of E. coli JM101 cells,several hundred recombinant phage were obtained. Duplex RF DNA wasprepared from twelve recombinant, phage-carrying clones. The RF DNA (0.1ug) was cleaved with EcoRI, (1 unit, 1 hour, 37° C.), end-labeled with³²P-dATP and Klenow polymerase, and re-digested with BamHI (1 unit, 1hour, 37° C.). The EcoRI and BamHI sites span the SmaI site. Therefore,clones containing the 260 bp MboI fragment could be identified asyielding a labelled 270 bp fragment after electrophoresis on 6%poly-acrylamide gels and autoradiography. Four of the twelve clonescarried this fragment. The orientation of the insert was determined bydigestion of the EcoRI-cleaved, end-labeled RF DNA (0.1 ug) with HinfI(1 unit, 1 hour, 37° C.). HinfI cleaves the 260 bp MboI fragment once 99bp from the 3′ end of the fragment and again 42 bp from the end nearestthe NOS coding region. Two clones of each orientation were obtained. Oneclone, digested as M-2 as shown in FIG. 8, contained the 260 bp fragmentwith the EcoRI site at the 3′ end of the fragment. M-2 RF DNA wasprepared using the procedures of Messing, et al 1981.

Example 7 Creation of pMON75 and pMON76

Fifty ug of M-2 RF DNA (described in Example 6) were digested with 50units of EcoRI and 50 units of BamHI for 2 hours at 37°. The 270 bpfragment 1(ug) was purified using agarose gel and NA-45 membrane.Plasmid pMON58 (described in Example 4) was digested with EcoRI andBamHI (50 ug, 50 units each, 2 hours, 37° C.) and the 1300 bp fragmentwas purified using NA-45 membrane. The 270 bp EcoRI-BamHI (0.1 ug) and1300 bp EcoRI-BamHI (0.5 ug) fragments were mixed, treated with T4 DNAligase (2 units) for 12 hours at 14° C. After heating at 70° C. for 10minutes to inactivate the ligase, the mixture was treated with EcoRI (10units) for 1 hour at 37° C., then heated to 70° C. for 10 minutes toinactivate the EcoRI. This completed the assembly of a chimericNOS-NPTII-NOS gene on a 1.6 kb fragment, as shown on FIG. 9.

Plasmid pMON38 is a clone of the pTiT37 HindIII-23 fragment inserted inthe HindIII site of pBR327 (Soberon et al., 1980). pMON38 DNA (20 ug)was digested with EcoRI (20 units, 2 hours, 37° C.) and calf alkalinephosphatase (0.2 units, 1 hour, 37° C.). The pMON38 DNA reaction wasextracted with phenol, precipitated with ethanol, dried and resuspendedin 20 ul of 10 mM Tris-HCl, 1 mM EDTA, pH 8.

0.2 ug of the cleaved pMON38 DNA was added to the chimeric gene mixturedescribed above. The mixture was treated with T4 DNA ligase (4 units, 1hour, 22° C.) and mixed with Rb chloride-treated E. coli C600 recA56cells to obtain transformation. After plating with selection forampicillin-resistant (200 ug/ml) colonies, 63 potential candidates wereobtained. Alkaline mini-preps of plasmid DNA were made from 12 of theseand screened by restriction endonuclease digestion for the properconstructs. Plasmid DNA's that contained a 1.5 kb EcoRI fragment and anew BglII site were digested with BamHI to determine the orientation ofthe 1.5 kb EcoRI fragment. One of each insert orientation was picked.One plasmid was designated pMON75 and the other pMON76, as shown in FIG.9. DNA from these plasmids were prepared as described in previousexamples.

Example 8 Creation of plasmids pMON128 and pMON129

The 1. 5 kb EcoRI fragment was excised by EcoRI digestion from eitherpMON75 or pMON76 and purified after agarose gel electrophoresis asdescribed in previous examples. Five ug of DNA from plasmid pMON120 wasdigested with EcoRI and treated with calf alkaline phosphatase. Afterphenol deproteinization and ethanol precipitation, the EcoRI-cleavedpMON120 linear DNA was mixed with 0.5 ug of the 1.5 kb EcoRI chimericgene fragment. The mixture was treated with 2 units of T4 DNA ligase for1 hour at 22° C. After transformation of E. coli cells and (Maniatis, etal., 1982) selection of colonies resistant to spectinomycin (50 ug/ml),several thousand colonies appeared. Six of these were picked, grown, andplasmid mini-preps made. The plasmid DNA's were digested with EcoRI tocheck for the 1.5 kb chimeric gene insert and with BamHI to determinethe orientation of the insert. BamHI digestion showed that in pMON128the chimeric gene was transcribed in the same direction as the intactnopaline synthase gene of pMON120. The orientation of the insert inpMON129 was opposite that in pMON128; the appearance of an additional1.5 kb BamHI fragment in digests of pMON129 showed that plasmid pMON129carried a tandem duplication of the chimeric NOS-NPTII-NOS gene, asshown in FIG. 10.

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What is claimed is:
 1. A chimeric plant-expressible gene, said genecomprising in the 5′ to 3′ direction: (a) a promoter region derived froma gene that is naturally expressed in a plant cell and that is capableof effecting mRNA transcription in the selected plant cell to betransfonned, operably linked to (b) a structural DNA sequence encoding apolypeptide that permits the selection of transformed plant cellscontaining said chimeric gene by rendering said transformed plant cellsresistant to an amount of an antibiotic that would be toxic tonon-transformed plant cells, operably linked to (c) a non-translatedregion of a gene naturally expressed in plant cells, said regionencoding a signal sequence for polyadenylation of mRNA.
 2. The gene ofclaim 1 in which the polypeptide renders transformed plant cellsresistant to an amount of an aminoglycoside antibiotic that would betoxic to non-transformed plant cells.
 3. The gene of claim 2 in whichthe polypeptide is a neomycin phosphotransferase polypeptide.
 4. Thegene of claim 1 in which the non-translated region is selected from agene from the group consisting of the genes of the T-DNA region ofAgrobacterium tumefaciens.
 5. The gene of claim 4 in which thenon-translated region is from the nopaline synthase gene ofAgrobacterium tumefaciens.
 6. The gene of claim 3 wherein saidpolypeptide is a neomycin phosphotransferase I polypeptide.
 7. The geneof claim 3 wherein said polypeptide is a neomycin phosphotransferase IIpolypeptide.
 8. A chimeric gene capable of expressing a polypeptide inplant cells comprising in sequence (a) a promoter region from a genewhich is naturally expressed in plant cells; (b) a 5′ non-translatedregion; (c) a structural coding sequence encoding a neomycinphosphotransferase polypeptide; and (d) a 3′ non-translated region of agene naturally expressed in plant cells, said region encoding a signalsequence for polyadenylation of mRNA.
 9. The gene of claim 8 in whichthe 3′ non-translated region is selected from a gene from the groupconsisting of the genes of the T-DNA region of Agrobacteriumtumefaciens.
 10. The gene of claim 9 in which the non-translated regionis from the nopaline synthase gene of Agrobacterium tumefaciens.
 11. Thegene of claim 8 wherein said polypeptide is a neomycinphosphotransferase I polypeptide.
 12. The gene of claim 8 wherein saidpolypeptide is a neomycin phosphotransferase II polypeptide.
 13. Amicroorganism containing a chimeric gene of claim
 1. 14. A microorganismcontaining a chimeric gene of claim
 8. 15. A culture of microorganismsof claim
 13. 16. A culture of microorganisms of claim
 14. 17. Theculture of claim 15 in which the microorganism is E. coli.
 18. Theculture of claim 15 in which the microorganism is Agrobacteriumtumefaciens.
 19. A plant cell susceptible to infection withAgrobacterium tumefaciens which contains and expresses a chimeric geneof claim
 1. 20. A plant cell susceptible to infection with Agrobacteriumtumefaciens which contains and expresses a chimeric gene of claim
 8. 21.Plant tissue comprising plant cells susceptible to infection withAgrobacierieim tumefaciens which contain and express a chimeric gene ofclaim
 1. 22. Plant tissue comprising plant cells susceptible toinfection with Agrobacterium tumefaciens which contain and express achimeric gene of claim
 8. 23. Plant tissue as in either claim 21 or 22wherein said tissue comprises undifferentiated tissue.